Metabolism is responsible for many life sustaining chemical processes that support cellular function through molecular and energetic transformations. Numerous pathways have evolved to sustain cellular bioenergetics and their balance can be critical for normal development and aging. Conversely, metabolic perturbations or dysfunctions are often implicated in numerous diseases, including obesity, diabetes, cancer, cardiovascular and neurodegenerative disorders. Accordingly, the ability to monitor subcellular functional and structural changes associated with metabolism can be essential for understanding tissue development and disease progression. However, established techniques are often either destructive or require the use of exogenous agents.
Generally, metabolic responses can be highly dynamic and heterogeneous both temporally and spatially, and this inherent heterogeneity can impact disease development or response to treatment significantly. Traditional imaging tools for assessing metabolic activity in vivo typically require addition of exogenous agents and can often have limited resolution and sensitivity. More sensitive, quantitative metabolic assays, such as those based on mass spectrometry and carbon labeling, cannot be readily performed within living cells and require cell and tissue homogenization. Therefore, such techniques can have limited capabilities for capturing dynamic or heterogeneous aspects of metabolic responses.
High resolution fluorescence imaging based approaches that rely on exogenous fluorescent probes can be sensitive to mitochondrial membrane potential or target specific cellular organelles or proteins, and can, therefore, overcome the latter limitations. However, such techniques often require cellular manipulations and can be confounded by artifacts related to the distributions of the fluorophores, especially in more complex, three-dimensional (3D) tissues. Therefore, quantitative, high-resolution, label-free techniques for examining metabolic processes, non-invasively and in vivo in 3D tissues, are needed to assist with characterizing and elucidating the role of different metabolic pathways in disease development, and as potential therapeutic targets.
Additionally, mitochondria can undergo trafficking, fusion, and fission, creating continuously changing networks to support mitochondrial function and accommodate cellular homeostasis. Aberrant mitochondrial dynamics and the corresponding changes in mitochondrial organization are increasingly associated with a variety of human pathologies, including neurodegenerative, metabolic, cardiovascular and neoplastic diseases. Many conventional methods for investigating mitochondrial morphology are invasive relying, for example, on scanning electron microscopy, mitochondria-specific dyes, or genetically engineered expression of fluorescent proteins. Accordingly, there is a need for improved methods and systems for assessing mitochondrial organization and dynamics.